Effects of household washing on bacterial load and removal of Escherichia coli from lettuce and "ready-to-eat" salads.

Customer demands for fresh salads are increasing, but leafy green vegetables have also been linked to food-borne illness due to pathogens such as Escherichia coli O157:H7. As a safety measure, consumers often wash leafy vegetables in water before consumption. In this study, we analyzed the efficiency of household washing to reduce the bacterial content. Romaine lettuce and ready-to-eat mixed salad were washed several times in flowing water at different rates and by immersing the leaves in water. Lettuce was also inoculated with E. coli before washing. Only washing in a high flow rate (8 L/min) resulted in statistically significant reductions (p < .05), "Total aerobic count" was reduced by 80%, and Enterobacteriaceae count was reduced by 68% after the first rinse. The number of contaminating E. coli was not significantly reduced. The dominating part of the culturable microbiota of the washed lettuce was identified by rRNA 16S sequencing of randomly picked colonies. The majority belonged to Pseudomonadaceae, but isolates from Enterobacteriaceae and Staphylococcaceaceae were also frequently found. This study shows the inefficiency of tap water washing methods available for the consumer when it comes to removal of bacteria from lettuce. Even after washing, the lettuce contained high levels of bacteria that in a high dose and under certain circumstances may constitute a health risk.

Antihypertensive activity of blueberries fermented by Lactobacillus plantarum DSM 15313 and effects on the gut microbiota in healthy rats.

BACKGROUND & AIMS: The aim of the present animal study was to examine the anti-hypertensive capacity of two probiotic products combining blueberries and the tannase producing probiotic bacteria Lactobacillus plantarum DSM 15313 and to investigate if such an effect is linked to a change in the gut microbiota. METHODS: Male Sprague Dawley rats were randomly divided into six groups of nine each. Three groups of the animals were treated with N(G)-nitro-L-arginine methyl ester (L-NAME) in the drinking water (40 mg/L) to induce a hypertensive state, and the other three groups were not treated with L-NAME (healthy rats). Two blueberry products differing in their phenolic acid content were tested and each rat received 2 g/day of the fermented blueberry powders for 4 weeks. The effects of the study products on the blood pressure, blood lipids, inflammatory markers, organ weights as well as caecal microbiota of the healthy (non-L-NAME-treated) rats were analyzed. RESULTS: After four weeks, healthy rats consuming freeze dried fermented blueberries with probiotics had a significant reduction in blood pressure compared to the control rats. In rats with L-NAME induced hypertension there was a significant reduction of the blood pressure after two weeks treatment. The probiotic product with a higher content of phenolic acids reduced ALAT in the healthy rats. Furthermore, ingestion of the probiotic blueberry products resulted in changes of the gut microbiota in the healthy rats. CONCLUSIONS: Blueberries fermented with the tannase producing bacteria L. plantarum DSM 15313 have anti-hypertensive properties and may reduce the risk for cardiovascular diseases.

Intake of Blueberry Fermented by Lactobacillus plantarum Affects the Gut Microbiota of L-NAME Treated Rats.

Prebiotics, probiotics, or synbiotics can be used as means to regulate the microbiota to exert preventative or beneficial effects to the host. However, not much is known about the effect of the gut microbiota on hypertension which is a major risk factor of cardiovascular disease and also a symptom of the metabolic syndrome. The N(G)-nitro-L-arginine methyl ester (L-NAME) induced hypertensive rats were used in order to test the effect of a synbiotic dietary supplement of Lactobacillus plantarum HEAL19 either together with fermented blueberry or with three phenolic compounds synthesized during fermentation. The experimental diets did not lower the blood pressure after 4 weeks. However, the fermented blueberries together with live L. plantarum showed protective effect on liver cells indicated by suppressed increase of serum alanine aminotransferase (ALAT) levels. The diversity of the caecal microbiota was neither affected by L-NAME nor the experimental diets. However, inhibition of the nitric oxide synthesis by L-NAME exerted a selection pressure that led to a shift in the bacterial composition. The mixture of fermented blueberries with the bacterial strain altered the caecal microbiota in different direction compared to L-NAME, while the three phenolic compounds together with the bacteria eliminated the selection pressure from the L-NAME.

Green tea powder and Lactobacillus plantarum affect gut microbiota, lipid metabolism and inflammation in high-fat fed C57BL/6J mice.

BACKGROUND: Type 2 diabetes is associated with obesity, ectopic lipid accumulation and low-grade inflammation. A dysfunctional gut microbiota has been suggested to participate in the pathogenesis of the disease. Green tea is rich in polyphenols and has previously been shown to exert beneficial metabolic effects. Lactobacillus plantarum has the ability to metabolize phenolic acids. The health promoting effect of whole green tea powder as a prebiotic compound has not been thoroughly investigated previously. METHODS: C57BL/6J mice were fed a high-fat diet with or without a supplement of 4% green tea powder (GT), and offered drinking water supplemented with Lactobacillus plantarum DSM 15313 (Lp) or the combination of both (Lp + GT) for 22 weeks. Parameters related to obesity, glucose tolerance, lipid metabolism, hepatic steatosis and inflammation were examined. Small intestinal tissue and caecal content were collected for bacterial analysis. RESULTS: Mice in the Lp + GT group had significantly more Lactobacillus and higher diversity of bacteria in the intestine compared to both mice in the control and the GT group. Green tea strongly reduced the body fat content and hepatic triacylglycerol and cholesterol accumulation. The reduction was negatively correlated to the amount of Akkermansia and/or the total amount of bacteria in the small intestine. Markers of inflammation were reduced in the Lp + GT group compared to control. PLS analysis of correlations between the microbiota and the metabolic variables of the individual mice showed that relatively few components of the microbiota had high impact on the correlation model. CONCLUSIONS: Green tea powder in combination with a single strain of Lactobacillus plantarum was able to promote growth of Lactobacillus in the intestine and to attenuate high fat diet-induced inflammation. In addition, a component of the microbiota, Akkermansia, correlated negatively with several metabolic parameters known to be risk factors for the development of type 2 diabetes.

Mucosa-associated bacteria in two middle-aged women diagnosed with collagenous colitis.

AIM: To characterize the colon microbiota in two women histologically diagnosed with collagenous colitis using a culture-independent method. METHODS: Biopsies were taken from the ascending colon and the total DNA was extracted. Universal bacterial primers were used to amplify the bacterial 16S rRNA genes. The amplicons were then cloned into competent Escherichia coli cells. The clones were sequenced and identified by comparison to known sequences. RESULTS: The clones could be divided into 44 different phylotypes. The microbiota was dominated by Firmicutes and Bacteroidetes. Seven phylotypes were found in both patients and constituted 47.5% of the total number of clones. Of these, the most dominating were clones similar to Bacteroides cellulosilyticus, Bacteroides caccae, Bacteroides thetaiotaomicron, Bacteroides uniformis and Bacteroides dorei within Bacteroidetes. Sequences similar to Faecalibacterium prausnitzii and Clostridium citroniae were also found in both patients. CONCLUSION: A predominance of potentially pathogenic Bacteroides spp., and the presence of clones showing similarity to Clostridium clostridioforme were found but the overall colon microbiota showed similarities to a healthy one. Etiologies for collagenous colitis other than an adverse bacterial flora must also be considered.

Lactobacillus plantarum 299v does not reduce enteric bacteria or bacterial translocation in patients undergoing colon resection.

BACKGROUND: Probiotics may exert beneficial effects in the gastrointestinal tract. This randomized trial investigated the effect of the probiotic Lactobacillus plantarum 299v on the intestinal load of potentially pathogenic bacteria, bacterial translocation, and cell proliferation in elective colon surgery. METHODS: Seventy-five patients were randomized to pre- and postoperative oral intake of Lactobacillus plantarum 299v or placebo. Rectal swabs and mucosal biopsies were taken before the start of intake, after 1 week, at surgery, and after 6 days, weeks, and months. Viable counts were quantified for clostridia, Enterobacteriaceae, Gram-negative anaerobes, and lactobacilli. Bacterial translocation was determined by the analysis of bacterial DNA genes in mesenteric lymph nodes. Ki-67 was used as a marker of cell proliferation in normal mucosa and tumor. RESULTS: Lactobacillus plantarum 299v was given without adverse effects. Lactobacillus plantarum 299v as well as Enterobacteriaceae and Gram-negative anaerobes increased in the colon 1 week after the administration of Lactobacillus plantarum 299v. There were no significant differences between patients receiving Lactobacillus plantarum 299v and placebo in the incidence of bacterial translocation (27 vs. 13%) and postoperative complications (16 vs. 31%). CONCLUSIONS: Lactobacillus plantarum 299v was established in the intestine, but no inhibitory effect on enteric bacteria, bacterial translocation, or postoperative complications was found. The mechanism behind the protective effects of probiotics found in animal and some human studies remain elusive and require further explorations. No adverse effects were recorded after the administration of high doses of Lactobacillus plantarum 299v.

The Lactobacillus flora in vagina and rectum of fertile and postmenopausal healthy Swedish women.

BACKGROUND: Lactobacillus species are the most often found inhabitants of vaginal ecosystem of fertile women. In postmenopausal women with low oestrogen levels, Lactobacillus flora is diminishing or absent. However, no studies have been performed to investigate the correlation between oestrogen levels and the lactobacilli in the gut. The aim of the present study was to investigate the relation in healthy women between vaginal and rectal microbial flora as well as possible variations with hormone levels. METHODS: Vaginal and rectal smears were taken from 20 healthy fertile women, average 40 years (range 28-49 years), in two different phases of the menstrual cycle, and from 20 postmenopausal women, average 60 years (range 52-85 years). Serum sex hormone levels were analyzed. Bacteria from the smears isolated on Rogosa Agar were grouped by Randomly Amplified Polymorphic DNA and identified by multiplex PCR and partial 16S rRNA gene sequencing. RESULTS: Lactobacillus crispatus was more often found in the vaginal flora of fertile women than in that of postmenopausal (p = 0.036). Fifteen of 20 fertile women had lactobacilli in their rectal smears compared to 10 postmenopausal women (p = 0.071). There was no correlation between the number of bacteria in vagina and rectum, or between the number of bacteria and hormonal levels. Neither could any association between the presence of rectal lactobacilli and hormonal levels be found. CONCLUSION: Lactobacillus crispatus was more prevalent in the vaginal flora of fertile women, whereas the Lactobacillus flora of rectum did not correlate to the vaginal flora nor to hormonal levels.

Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates.

Molecular typing of Klebsiella species has become important for monitoring dissemination of ß-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)(5)- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)(5) and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)(5) and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)(5) and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.

Reduced diversity in the early fecal microbiota of infants with atopic eczema.

BACKGROUND: It might be that early intestinal colonization by bacteria in westernized infants fails to give rise to sufficient immune stimulation to support maturation of regulatory immune mechanisms. OBJECTIVE: The purpose of the present study was to characterize the very early infantile microbiota by using a culture-independent approach and to relate the colonization pattern to development of atopic eczema in the first 18 months of life. METHODS: Fecal samples were collected from 35 infants at 1 week of age. Twenty infants were healthy, and 15 infants were given diagnoses of atopic eczema at the age of 18 months. The fecal microbiota of the infants was compared by means of terminal restriction fragment length polymorphism (T-RFLP) and temporal temperature gradient gel electrophoresis (TTGE) analysis of amplified 16S rRNA genes. RESULTS: By means of T-RFLP analysis, the median number of peaks, Shannon-Wiener index, and Simpson index of diversity were significantly less for infants with atopic eczema than for infants remaining healthy in the whole group and for the Swedish infants when AluI was used for digestion. The same was found when TTGE patterns were compared. In addition, TTGE analysis showed significantly less bands and lower diversity indices for the British atopic infants compared with those of the control subjects. CONCLUSION: There is a reduced diversity in the early fecal microbiota of infants with atopic eczema during the first 18 months of life.

Ileal pelvic pouch microbiota from two former ulcerative colitis patients, analysed by DNA-based methods, were unstable over time and showed the presence of Clostridium perfringens.

OBJECTIVE: Ileal pouch anal anastomosis (IPAA) is the preferred method for restorative surgery in patients with ulcerative colitis who have to undergo proctocolectomy. The most common complication is pouchitis and several studies have pointed to the microbiota of the pouch as being a risk factor. The aim of this study was to follow the development of the bacterial microbiota in pouches during the first year. MATERIAL AND METHODS: Terminal restriction fragment length polymorphism (T-RFLP) combined with cloning and sequencing was used to identify the most predominant bacteria on the different sampling occasions. A total of 274 clones were grouped by T-RFLP and clones from each group were selected for sequencing and identified by comparison with known sequences. RESULTS: Differences in T-RFLP profiles and clone libraries were found between the patients, and also in changes apparent in each patient at different time-points. The main bacterial groups in the pouches resembled those of the normal colonic microbiota, with a predominance of the clostridia clusters XIVa and IV, Bacteroides and Enterobacteriaceae. Exceptions were clones with sequences resembling those of the Clostridium perfringens group, in both patients and on all sampling occasions, and the dominance of clones resembling Turicibacter in one of the patients at the time of pouch construction. CONCLUSIONS: The pouch microbiota showed similarities to the normal colon microbiota except for the presence of clones with sequences resembling those of the C. perfringens group and Turicibacter. The bacterial composition differed between the two patients and the microbiota changed with time, suggesting that the composition is not stable during the first year.

Adhesive capability of Lactobacillus plantarum 299v is important for preventing bacterial translocation in endotoxemic rats.

The preventive effect of the probiotic Lactobacillus plantarum 299v on bacterial translocation (BT) and the role of adhesion were studied in septic rats. Five groups of rats were pretreated as follows: negative and positive control groups received regular drinking water; the oatmeal group received drinking water mixed with oatmeal; the Lp 299v group received drinking water mixed with oatmeal containing 10(9) colony-forming units (CFU) L. plantarum 299v/ml; the Lp 299v-adh(-) group received drinking water with oatmeal containing 10(9) CFU/ml of modified L. plantarum 299v (L. plantarum 299v-adh(-)) lacking adhesive properties to enterocytes. On day 8, all rats except the negative control group were given lipopolysaccharide (LPS) intraperitoneally. After 24 h, mesenteric lymph node (MLN), liver and ileum were harvested for culture. Incidence of BT after LPS challenge was 25% and 88% in MLN and liver, respectively. BT increased to 75% in MLN and 100% in liver of endotoxemic rats pretreated with oatmeal. Pretreatment with L. plantarum 299v reduced BT to 0% and 12% in MLN and liver, respectively. L. plantarum 299v-adh(-) did not prevent BT to MLN. Flow cytometry revealed reduced adherence of these bacteria to intestinal epithelial cells compared to L. plantarum 299v. Thus, L. plantarum 299v prevents BT in septic rats, an effect probably dependent on bacterial adherence to the intestinal mucosa. Further, our findings indicate that oatmeal (prebiotics) without probiotics does not prevent BT during sepsis.

Multiple displacement amplification of DNA from human colon and rectum biopsies: bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis.

Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis; the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens.

DNA based classification of food associated Enterobacteriaceae previously identified by Biolog GN Microplates.

Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons. Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods. In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae. The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis. All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene. Selected strains were analysed by ribotyping and partial 16S rDNA sequencing. The type strains were differentiated into 10 different TTGE groups. Two of the groups contained two type strains. Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern. The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains. Rahnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3. The grouping of Rahnella strains was supported by ribotyping and phylogenetic analysis. TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.

The bacterial flora of fresh and chill-stored pork: analysis by cloning and sequencing of 16S rRNA genes.

The composition of the initial bacterial flora of pork and the development of the flora after storage at +4 degrees C for 4 days were analysed by amplification, cloning and sequencing of 16S rDNA. A total of 122 clones were obtained, with lengths of > or =400 nucleotides and > or =95% similarity to database sequences. Nineteen clones were similar to sequences in database not assigned to any genera. Fourteen different genera were represented in clones from fresh meat, with 36.5% of the clones most resembling Acinetobacter and 17.3% resembling Staphylococcus and Macrococcus. After storage, the clones were composed of six different genera, with 44.3% resembling Pseudomonas, 17.1% resembling Aeromonas and only 14.3% resembling Acinetobacter. This study shows that the overall pattern of the initial and chill-stored pork flora, as shown by a molecular approach, was in agreement with results obtained in previous studies using traditional cultivation methods.